Brendon Parsons

A Direct Phenotypic Comparison of siRNA Pools and Multiple Individual Duplexes in a Functional Assay

Background RNAi is a prominent tool for the identification of novel regulatory elements within complex cellular pathways. In invertebrates, RNAi is a relatively straightforward process, where large double-stranded RNA molecules initiate sequence-specific transcript destruction in target cells. In contrast, RNAi in mammalian cell culture assays requires the delivery of short interfering RNA duplexes to target cells. Due to concerns over off-target phenotypes and extreme variability in duplex efficiency, investigators typically deliver and analyze multiple duplexes per target. Currently, duplexes are delivered and analyzed either individually or as a pool of several independent duplexes. A choice between experiments based on siRNA pools or multiple individual duplexes has considerable implications for throughput, reagent requirements and data analysis in genome-wide surveys, yet there are relatively few data that directly compare the efficiency of the two approaches. Methodology/Principal Findings To address this critical issue, we conducted a direct comparison of siRNA pools and multiple single siRNAs that target all human phosphatases in a robust functional assay. We determined the frequency with which both approaches uncover loss-of-function phenotypes and compared the phenotypic severity for siRNA pools and the constituent individual duplexes. Conclusions/Significance Our survey indicates that screens with siRNA pools have several significant advantages over identical screens with the corresponding individual siRNA duplexes. Of note, we frequently observed greater phenotypic penetrance for siRNA pools than for the parental individual duplexes. Thus, our data indicate that experiments with siRNA pools have a greater likelihood of generating loss-of-function phenotypes than individual siRNA duplexes.

The Drosophila Platelet-derived Growth Factor and Vascular Endothelial Growth Factor-Receptor Related (Pvr) Protein Ligands Pvf2 and Pvf3 Control Hemocyte Viability and Invasive Migration

Background: PDGF- and VEGF-related factor (Pvr) ligands are implicated in the establishment and dispersal of Drosophila embryonic hemocytes. Results: Hemocyte-restricted Pvr pathway activation in pvf2–3 mutants is sufficient for hemocyte survival and non-invasive migration, whereas epithelial-specific Pvf expression is required for hemocyte invasive migration. Conclusion: Pvfs are required for invasion, but not directional guidance of hemocytes. Significance: We uncover a novel role for Pvfs in hemocyte trans-epithelial migration. Vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF) family members are essential and evolutionary conserved determinants of blood cell development and dispersal. In addition, VEGFs are integral to vascular growth and permeability with detrimental contributions to ischemic diseases and metastatic cancers. The PDGF/VEGF-receptor related (Pvr) protein is implicated in the migration and trophic maintenance of macrophage-like hemocytes in Drosophila melanogaster embryos. pvr mutants have a depleted hemocyte population and a breakdown in hemocyte distribution. Previous studies suggested redundant functions for the Pvr ligands, Pvf2 and Pvf3 in the regulation of hemocyte migration, proliferation, and size. However, the precise roles that Pvf2 and Pvf3 play in hematopoiesis remain unclear due to the lack of available mutants. To determine Pvf2 and Pvf3 functions in vivo, we generated a genomic deletion that simultaneously disrupts Pvf2 and Pvf3. From our studies, we identified contributions of Pvf2 and Pvf3 to the Pvr trophic maintenance of hemocytes. Furthermore, we uncovered a novel role for Pvfs in invasive migrations. We showed that Pvf2 and Pvf3 are not required for the directed migration of hemocytes, but act locally in epithelial cells to coordinate trans-epithelial migration of hemocytes. Our findings redefine Pvf roles in hemocyte migration and highlight novel Pvf roles in hemocyte invasive migration. These new parallels between the Pvr and PDGF/VEGF pathways extend the utility of the Drosophila embryonic system to dissect physiological and pathological roles of PDGF/VEGF-like growth factors.

Detection, Characterization, and Typing of Shiga Toxin-Producing Escherichia coli

Shiga toxin-producing Escherichia coli (STEC) are responsible for gastrointestinal diseases reported in numerous outbreaks around the world. Given the public health importance of STEC, effective detection, characterization and typing is critical to any medical laboratory system. While non-O157 serotypes account for the majority of STEC infections, frontline microbiology laboratories may only screen for STEC using O157-specific agar-based methods. As a result, non-O157 STEC infections are significantly under-reported. This review discusses recent advances on the detection, characterization and typing of STEC with emphasis on work performed at the Alberta Provincial Laboratory for Public Health (ProvLab). Candidates for the detection of all STEC serotypes include chromogenic agars, enzyme immunoassays (EIA) and quantitative real time polymerase chain reaction (qPCR). Culture methods allow further characterization of isolates, whereas qPCR provides the greatest sensitivity and specificity, followed by EIA. The virulence gene profiles using PCR arrays and stx gene subtypes can subsequently be determined. Different non-O157 serotypes exhibit markedly different virulence gene profiles and a greater prevalence of stx1 than stx2 subtypes compared to O157:H7 isolates. Finally, recent innovations in whole genome sequencing (WGS) have allowed it to emerge as a candidate for the characterization and typing of STEC in diagnostic surveillance isolates. Methods of whole genome analysis such as single nucleotide polymorphisms and k-mer analysis are concordant with epidemiological data and standard typing methods, such as pulsed-field gel electrophoresis and multiple-locus variable number tandem repeat analysis while offering additional strain differentiation. Together these findings highlight improved strategies for STEC detection using currently available systems and the development of novel approaches for future surveillance.

Cellular immune defenses of Drosophila melanogaster.

Drosophila melanogaster is a widely used model for the characterization of blood cell development and function, with an array of protocols for the manipulation and visualization of fixed or live cells in vitro or in vivo. Researchers have deployed these techniques to reveal Drosophila hemocytes as a remarkably versatile cell type that engulfs apoptotic corpses; neutralizes invading parasites; seals epithelial wounds; and deposits extracellular matrix proteins. In this review, we will discuss the key features of Drosophila hemocyte development and function, and identify similarities with vertebrate counterparts.

A Deregulated Intestinal Cell Cycle Program Disrupts Tissue Homeostasis without Affecting Longevity in Drosophila

Background: We know comparatively little about the impact of deregulated intestinal proliferation on an otherwise normal animal. Results: Modified progression through the cell cycle disrupted homeostasis without affecting longevity. Conclusion: Disrupted intestinal homeostasis alone does not shorten the life span of an adult fly. Significance: These questions are of considerable importance, given the relationship between proliferation, dysplasia, aging, and death. Recent studies illuminate a complex relationship between the control of stem cell division and intestinal tissue organization in the model system Drosophila melanogaster. Host and microbial signals drive intestinal proliferation to maintain an effective epithelial barrier. Although it is widely assumed that proliferation induces dysplasia and shortens the life span of the host, the phenotypic consequences of deregulated intestinal proliferation for an otherwise healthy host remain unexplored. To address this question, we genetically isolated and manipulated the cell cycle programs of adult stem cells and enterocytes. Our studies revealed that cell cycle alterations led to extensive cell death and morphological disruptions. Despite the extensive tissue damage, we did not observe an impact on longevity, suggesting a remarkable degree of plasticity in intestinal function.

Enteropathogen detection in children with diarrhoea, or vomiting, or both, comparing rectal flocked swabs with stool specimens: an outpatient cohort study

BACKGROUND Enteropathogen detection traditionally relies on diarrhoeal stool samples, but these are inconvenient to collect if they are not immediately available, leading to suboptimum return rates of samples and delayed or missed diagnostic opportunities. We sought to compare the enteropathogen yields of rectal swabs and stool specimens in children with diarrhoea or vomiting, or both. METHODS The Alberta Provincial Pediatric EnTeric Infection TEam (APPETITE) did a study in three outpatient cohorts in Calgary and Edmonton (AB, Canada)-children enrolled in the Pediatric Emergency Research Canada emergency departments, children receiving routine vaccinations at a Calgary health clinic, and symptomatic children who met criteria for treatment at home. Eligible participants were children younger than 18 years, with at least three episodes of vomiting or diarrhoea in the preceding 24 h and fewer than 7 days of symptoms. After excluding those enrolled within the previous fortnight, unable to follow-up, or having psychiatric illness, neutropenia, or requiring emergent care, we attempted to collect rectal swabs and stool from all participants. Specimens were tested with the multianalyte assay Luminex xTAG Gastrointestinal Pathogen Panel, an in-house five-virus panel and bacterial culture. Primary outcomes were comparative yield (calculated as the proportion of submitted paired specimens only in which at least one pathogen was identified) and overall yield (which calculated the proportion of study participants in whom at least one pathogen was identified in all specimens, where unsubmitted specimens were analysed as negative). We used McNemars test to do pathogen-specific analyses, and generalised estimating equations (GEE) for the global (ie, any) pathogen analyses, with adjustments made for the presence of diarrhoea, location, and their interactions with specimen type. FINDINGS Between Dec 12, 2014, and Aug 31, 2016, we studied 1519 eligible participants, 1147 (76%) of whom provided stool specimens and 1514 (>99%) provided swab specimens. 871 (76%) of 1147 stool specimens and 1024 (68%) of 1514 swabs were positive for any pathogen (p<0·0001). Comparative yield adjusted odds ratios (ORs) for stool specimens relative to swabs were 1·24 (95% CI 1·11-1·38) in children with diarrhoea at presentation and 1·76 (1·47-2·11) in children without diarrhoea. GEE analysis identified an interaction between the presence of diarrhoea and specimen type (p=0·0011) and collection location (p=0·0078). In an overall yield analysis, pathogen yield was 57% (871 of 1519 children) for stool specimens and 67% (1024 of 1519 children) for rectal swabs, with an unadjusted OR of 0·65 (95% CI 0·59-0·72) for stool relative to swab. INTERPRETATION Rectal swabs should be done when enteropathogen identification and rapid detection are needed, appropriate molecular diagnostic technology is available, and a stool specimen is not immediately available. In view of their high yield, we urge that the recommendation against the use of rectal swabs as diagnostic specimens be reconsidered. FUNDING Alberta Innovates-Health Solutions Team Collaborative Research Innovation Opportunity.

High genetic variability of norovirus leads to diagnostic test challenges

BACKGROUND It is important to understand the diagnostic accuracy of multiplex panels such as the Luminex xTAG® Gastrointestinal Pathogen Panel (GPP) as they are increasingly employed for routine diagnostics worldwide. Recent evaluations in our laboratory identified lower detection rates of norovirus genogroup II (NoV GII) using GPP compared to our laboratory-developed RT-qPCR, Gastroenteritis Virus Panel (GVP). OBJECTIVES To characterize the cases of discordant NoV GII results between GPP and GVP and determine the sensitivity of the two assays for specific NoV GII genotypes. STUDY DESIGN We genotyped discordant NoV GII strains identified in stool samples or rectal swabs collected prospectively from a cohort of children with acute gastroenteritis between December 2014 and July 2016. The sensitivities of GVP and GPP for NoV GII were compared by analyses of GVP threshold cycle (Ct) and ten-fold serial dilutions of positive samples of various NoV GII genotypes. RESULTS All discordant samples (63/607) were NoV GII positive by GVP but negative by GPP. Twenty-two were successfully genotyped, fourteen of which were NoV GII genotype 2 (GII.2). The median Ct value of concordant positives was lower than that of discordant results (19.8 vs. 33.7; P<0.0001). GVP was 10 and at least 10,000-fold more sensitive than GPP in detecting NoV GII.3 and GII.2, respectively, but has similar sensitivity for NoV GII.4. Discordant GII.2 variant differed genetically from concordant GII.2 variants. CONCLUSIONS GPP has lower sensitivity to detect NoV GII.2 than GVP and its use may lead to undetected cases clinically, and an underestimation of NoV disease burden at the population level.

Diagnostic Interpretation Guidance for Pediatric Enteric Pathogens: A Modified Delphi Consensus Process

Background We sought to develop diagnostic test guidance definitions for pediatric enteric infections to facilitate the interpretation of positive test results in the era of multianalyte molecular diagnostic test platforms. Methods We employed a systematic, two-phase, modified Delphi consensus process consisting of three web-based surveys and an expert panel face-to-face meeting. In phase 1, we surveyed an advisory panel of North American experts to select pathogens requiring diagnostic test guidance definition development. In phase 2, we convened a 14-member expert panel to develop, refine, and select the final definitions through two web-based questionnaires interspersed with a face-to-face meeting. Both questionnaires asked panelists to rate the degree to which they agreed that if the definition is met the pathogen is likely to be causative of clinical illness. Results The advisory panel survey identified 19 pathogens requiring definitions. In the expert panel premeeting survey, 13 of the 19 definitions evaluated were rated as being highly likely (“agree” or “strongly agree”) to be responsible for acute gastroenteritis symptoms by ≥67% of respondent panel members. The definitions for the remaining six pathogens (Aeromonas, Clostridium difficile, Edwardsiella, nonenteric adenovirus, astrovirus, and Entamoeba histolytica) were indeterminate. After the expert panel meeting, only two of the modified definitions, C. difficile and E. histolytica/dispar, failed to achieve the a priori specified threshold of ≥67% agreement. Conclusions We developed diagnostic test guidance definitions to assist healthcare providers for 17 enteric pathogens. We identified two pathogens that require further research and definition development.

Gastroenteritis Severity: A Prospective Cohort Comparison of Children in Emergency Department and Home Settings

Abstract Background While nearly 2 million children are brought to emergency department (ED) annually due to vomiting and/or diarrhea from acute gastroenteritis (AGE), it is estimated that 90% of AGE cases do not seek medical care. We sought to determine whether the disease severity and enteropathogen burden of disease of children with AGE brought for ED care is different from those cared for at home. Methods Subjects were prospectively recruited by the APPETITE team in pediatric EDs in 2 urban centers and via HealthLink, a province-wide nurse telephone advice line. Eligibility criteria included: < 18 years old, AGE defined by ≥ 3 episodes of vomiting or diarrhea in the preceding 24 hours, and < 7 days of symptoms. The primary outcome was index encounter disease severity quantified using the modified Vesikari Scale (MVS) score. To eliminate the impact of the index encounter on the score we excluded the index ED visit and intervention from all calculations. Secondary objectives included the enteropathogen burden of disease. Two rectal swabs and stool were collected and tested for enteropathogens by enteric bacterial culture, Luminex xTAG GPP, and a 5-virus in-house RT-qPCR panel. Results Between December 9, 2014 and December 31, 2016, 1,623 participants were enrolled with 81.5% from the EDs. Median age was 20.1 months. Children who went to ED were less likely to have a family physician (62 vs. 82%, P < 0.001), more likely to have clinical dehydration (Clinical Dehydration Scale score 3 vs 1, P < 0.001) and vomiting (91 vs. 85%, P = 0.004), previously received IV fluids (4.1 vs. 0.7%, P = 0.001) or been admitted (5.4 vs. 1.3%, P = 0.002). The MVS score was similar between groups when the contribution of the index visit to the score was excluded (8.1 vs. 7.8, P = 0.15). Participants recruited in the ED were not significantly more likely to have bacterial pathogens (8.0 vs. 3.7%, P = 0.09) but were less likely to have viral pathogens identified (64.1 vs. 80.7, P < 0.001). Conclusion Children presenting for ED care had disease severity scores that were similar to children managed at home when the contribution of the index ED visit was accounted for. Viral pathogens were more common in AGE receiving care at home while those presenting to the ED and potentially have a clinically greater likelihood of having a bacterial enteropathogen. Disclosures All authors: No reported disclosures.

High Genetic Variability of Norovirus Leads to Diagnostic Test Challenges

Abstract Background It is important to understand the diagnostic accuracy of syndromic multiplex panels such as the Luminex xTAG® Gastrointestinal Pathogen Panel (GPP) as they are increasingly employed as routine diagnostic tests in laboratories worldwide. Recent evaluations in our laboratory identified lower detection rates of norovirus genogroup II (NoV GII) using the GPP as compared with our lab-developed RT-qPCR Gastroenteritis Virus Panel (GVP). This study is to characterize the NoV strains in samples with discordant NoV GII test results between GPP and GVP and determine the sensitivity of the two assays for specific NoV GII genotypes. Methods We genotyped all NoV GII strains with discordant test result in stool samples or rectal swabs collected prospectively from a cohort of children with acute gastroenteritis between December 2014 and July 2016. The sensitivity of GVP and GPP for NoV GII were compared by analyzing GVP threshold cycle (Ct) and using ten-fold serial dilutions of positive samples of various NoV GII genotypes. Results All discordant samples (11%; 63/607) tested positive for NoV GII by GVP but negative by GPP. Thirty-five percent (22/63) were successfully genotyped; 64% (14/22) of those were NoV GII genotype 2 (GII.2). The median Ct value of concordant positive was lower than those with discordant results (19.8 vs.. 33.7 respectively; P < 0.0001). GVP was 10-fold and at least 10,000-fold more sensitive than GPP in detecting NoV GII.3 and GII.2, respectively, but has similar sensitivity for NoV GII.4. The GII.2 variants with discordant test results differed genetically from the concordant GII.2 variants. Conclusion GPP has suboptimal sensitivity to detect NoV GII.2 and its use may lead to an underestimation of NoV disease burden with some cases not being detected. Disclosures All authors: No reported disclosures.